1. Field of the Invention
This invention pertains to the isolation, purification and characterization of four separate cockroach allergens identified as binding to IgE antibodies in humans, and thereby inducing, in sensitive individuals, an allergic or asthmatic response. Specifically, four cockroach allergens, designated according to the WHO allergens nomenclature as Bla g 2, Bla g 4, Bla g 5, and Bla g 6 are identified, Bla 4, 5 and 6 having never before been purified. Additionally, the nucleotide sequences for the genes responsible for expression of these allergens are given, and the amino acids sequences therefore. Recombinant expression of the allergens is achieved, leading to modification of the same.
2. Background of the Prior Art.
It has been known since at least 1964 that cockroaches (CR) produce potent allergens that can cause asthma and allergic respiratory disease. Numerous attempts over the past twenty years have been made to identify the important CR allergens that cause allergic (IgE) antibody responses in CR sensitive patients. CR extracts used for allergy testing comprise aqueous extracts of ground CR bodies. These extracts contain many other CR proteins in addition to the relevant allergens. The approach adopted by many research groups has been to use biochemical separation and purification techniques to isolate the allergens and to assess their allergenic importance by reactivity, in vitro and in vivo, with IgE antibody. Using this approach, some allergens have been characterized according to their molecule size and charge, and reactivity with IgE. Two of these were sufficiently well characterized to be included in the WHO allergens nomenclature, Bla g 1 and Bla g 2.
Notwithstanding these advances, the actual protein structures of the allergens were not known. Further, only the two allergens had been purified. It was not, therefore, possible to define the chemical structures of these allergens. This precludes developing information to determine antigenic sites in the molecules (IgE binding epitopes), or other information involved in the immune response. Additionally, isolation of the nucleotide sequence is a pre-requisite to its recombinant expression, and modification, through fusion technology, site-specific and site-directed mutation, and the like, so as to develop methods of treating and diagnosing CR allergies. The same has been successfully done in analagous areas, such as in connection with the house dust mite allergen Der P 2.
Isolation, characterization, and recombinant expression of the CR allergens can be used to improve methods of diagnosing CR allergy, which are currently confined to generic extract scratch testing, which precludes identifying the specific allergen responsible; developing new treatments for CR allergies and developing methods for controlling CR infestation.
Accordingly, it remains an object of those of ordinary skill in the art to purify, characterize, sequence and recombinantly express CR allergens.